Methods to facilitate diagnosis of normal pressure hydrocephalus

ABSTRACT

The present invention relates to methods for differentiating demential diseases comprising measuring the concentration of human lipocalin-type prostaglandin D synthase in a sample of a body fluid collected from a subject and kits for differentiating demential diseases comprising an antibody specific to human lipocalin-type prostaglandin D synthase.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP01/04811 which has an Internationalfiling date of Jun. 7, 2001, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to methods for differentiating dementialdiseases, more specifically methods for differentiating dementialdiseases comprising measuring a concentration of human lipocalin-typeprostaglandin D synthase (hereinafter periodically referred to as“L-PGDS”) in a sample of a body fluid collected from a subject anddifferentiation kits for use in the methods.

BACKGROUND ART

With the aging of society, there is an ongoing increase in the number ofpeople suffering from demential diseases. Demential diseases can beinduced by an enormous variety of etiologies. It is therefore verydifficult to make an exact differential diagnosis of such a disease, andno therapy has been established for many demential diseases. Amongdemential diseases, normal pressure hydrocephalus (hereinafter referredto as NPH) including symptomatic normal pressure hydrocephalus followingsubarachnoid hemorrhage or cerebral meningitis and idiopathic normalpressure hydrocephalus of unknown cause is known to be dramaticallyimproved by surgery (e.g. ventriculoperitoneal shunt). However, therecan be little expectation that surgery will have any effect in improvingsymptoms of demential diseases found in elderly people, such asAlzheimer's disease, Parkinson's disease and cerebrovascular dementia,or the symptoms of diffuse brain injury, characterized by cerebralatrophy and ventricular enlargement. Thus, it is important to be able todifferentiate at an early stage between NPH, which is treatable bysurgery, and dementia for which surgical treatment is not effective.However, it is difficult to make such differentiation.

A classic diagnostic method for determing suitability for surgeryinvolves continuously measuring a cerebrospinal pressure for a period ofone day via drainage from lumbar vertebra to cerebrospinal cavity, tothereby monitor a pressure wave of cerebrospinal fluid (Symon, L.,Dorsh, N. W. C., J. Neurosurg., 42: 258-273, 1975). However, this methodis often clinically difficult because it requires a patient to betreated at rest in a bed in a clean environment, using appropriateinstruments for continuous measurement and analysis and the like.Another method involves daily drainage of 40-50 ml of cerebrospinalfluid to assess improvements in symptoms, but this lacks reliability andcarries a risk of complication such as infection of a site subjected torepeated puncture. A method for differentiating between atrophicAlzheimer-type senile dementia and NPH by assaying amyloid-relatedprotein (α1-antichymotrypsin) in cerebral venous blood has beenreported, but is not widely used because it requires invasive collectionof cerebral venous blood and lacks effectiveness.

The rationale for monitoring a dynamic state of spinal fluid resides inthe pathophysiology of these diseases. However, it is to be noted thatmeasurement of a pressure wave of spinal fluid has been recentlyreplaced by methods made possible by remarkable advances in diagnosticimaging. A conventional standard method involves RI or CT cisternographywith a contrast medium injected into cerebrospinal cavity from lumbarvertebra to evaluate malabsorption of spinal fluid into circulation, butdiagnosis resulting from use of this method has been reported not toalways correlate properly to postoperative prognosis. A relatively newreport proposes evaluating a flow of spinal fluid in the aqueduct ofmidbrain using MRI images (Mase, M. et al., Current Treatment forHydrocephalus (Tokyo), 8:13-18, 1998). This is an attractivenon-invasive method, but is still under development and remainsineffective for some cases. One of the reasons that this method has notcome into widespread use is because it can be carried out in onlylimited facilities.

Among recent successful studies, a report proposes detecting NPH byassessing damage to nerve tissue based on neurofilament triplet protein(NFL) or glial fibrillary acidic protein (GFAP) in cerebrospinal fluid(Tullberg, M. et al., Neurology 60:1122-1127, 1998), but this method hasnot been made commercial yet.

As described above, surgery is effective for NPH, but there has not yetbeen developed any detection method for determining whether earlysurgical treatment of a patient suffering from a demential disease isrequired.

Prostaglandin D syntheses (PGDSs) include lipocalin type mainlylocalized in brain and hematopoietic organ type localized in spleen andmast cells, and the PGDS protein found in cerebrospinal fluid has beenidentified as lipocalin type. Lipocalin-type prostaglandin D synthase(L-PGDS) is an enzyme involved in biosynthesis of prostaglandin D₂ inthe central nerve system (CNS) of various mammals. This enzyme is mainlyproduced in cerebral leptomeninges and arachnoid membrane and secretedinto cerebrospinal fluid (hereinafter sometimes referred to as CSF).Recently, this L-PGDS has been shown to be identical with β-trace thatwas known to be present in abundance in CSF (Hoffmann A. et al., J.Neurochem., 61:451-456, 1993; Zahn M. et al., Neurosci. Let., 154:93-95,1993; Watanabe, K. et al., Biochem. Biophys. Res. Commun.,203:1110-1116, 1994). Studies have been devoted to clinical uses ofβ-trace in various diseases of the central nerve system because it is amain member of human CSF proteins. However, the involvement or role ofPGDS or L-PGDS in various demential diseases remains unexplaind.

An object of the present invention is therefor to provide a methodcapable of differentiating reliably and with a minimum of stress to apatient normal pressure hydrocephalus that was not detectable ordifficult to reliably detect by various conventional test means. Anotherobject of the present invention is to provide a kit for use in thedifferentiation method.

DISCLOSURE OF THE INVENTION

As a result of careful studies to attain the above objects, theinventors accomplished the present invention on the basis of the findingthat demential diseases can be differentiated by using L-PGDS levelsdetermined in a body fluid such as cerebrospinal fluid, blood or urineas an indicator.

Accordingly, the present invention provides a method for differentiatinga demential disease comprising measuring a concentration of humanlipocalin-type prostaglandin D synthase in a sample of a body fluidcollected from a subject. The present invention also provides a kit fordifferentiating a demential disease comprising an antibody specific tohuman lipocalin-type prostaglandin D synthase.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows assay results of L-PGDS levels in the cerebrospinal fluidof controls and patients with postsubarachnoid hemorrhage NPH andidiopathic NPH, cerebrovascular dementia, Parkinson's disease andAlzheimer's disease.

FIG. 2 shows assay results of L-PGDS levels in the cerebrospinal fluidof controls and patients with presenile NPH (y-NPH) and senile NPH(e-NPH) and dementia.

FIG. 3 shows assay results of neuron specific enolase (NSE) levels inthe cerebrospinal fluid of control subjests and in patients withpresenile NPH (y-NPH) and senile NPH (e-NPH) and dementia.

FIG. 4 shows assay results of S-100 protein levels in the cerebrospinalfluid of controls and patients with presenile NPH (y-NPH) and senile NPH(e-NPH) and dementia.

THE MOST PREFERRED EMBODIMENTS OF THE INVENTION

The sample assayed for L-PGDS in the present invention is a body fluidcollected from a subject, specifically cerebrospinal fluid, blood orurine, etc. The method for determining L-PGDS levels in the sample isnot specifically limited so far as it exactly reflects L-PGDS levels,such as immunoassays and enzymatic activity assays. However,immunoassays such as EIA, ELISA, RIA and FIA using a monoclonal orpolyclonal antibody specific to L-PGDS are preferred from the viewpointof the necessity of simultaneously assaying a large number of sampleswith convenience in the actual clinical field.

Among these immunoassays, especially preferred is sandwich ELISA usingan L-PGDS-specific monoclonal antibody such as antibodies produced byhybridoma cell lines 1B7 (FERM BP-5709), 7F5 (FERM BP-5711). 6F5 (FERMBP-5710), 9A6 (FERM BP-5712) and 10A3 (FERN BP-5713). For determinationby sandwich ELISA, an L-PGDS detection kit comprising the monoclonalantibody already established by the inventors can be used (WO97/16461).These cell lines have been internationally deposited under BudapestTreaty with the National Institute of Bioscience and Human-Technology ofthe Agency of Industrial Science and Technology (residing at 1-3,Higashi 1-Chome, Tsukuba-city, Ibaraki-prefecture, Japan).

In the present invention, NPH can be differentiated by using L-PGDSlevels determined by the means described above as an indicator incomparison with L-GDS levels in controls or L-PGDS levels in patientswith demential diseases other than NPH (e.g. Alzheimer's disease,Parkinson's disease, vascular dementia).

Demential diseases detected or differentiated by methods of the presentinvention include, for example, normal pressure hydrocephalus (NPH)including symptomatic normal pressure hydrocephalus followingsubarachnoid hemorrhage or cerebral meningitis and idiopathic normalpressure hydrocephalus of unknown cause. Methods of the presentinvention can be used to differentiate these NPHs from controls or otherdemential diseases such as vascular dementia, Alzheimer's disease orParkinson's disease.

NPH such as symptomatic NPH and idiopathic NPH is characterized bysignificantly low L-PGDS levels in cerebrospinal fluid as compared withthat of control subjects. NPH also shows significantly lower L-PGDSlevels in cerebrospinal fluid as compared with other demential diseasessuch as vascular dementia, Alzheimer's disease and Parkinson's disease.No significant difference was found between controls and patients withvascular dementia or Parkinson's disease.

L-PGDS levels in CSF were determined in NPH patients divided into twogroups, i.e. patients under 70 years of age (presenile NPH group) and ator more than 70 years of age (senile NPH group) to demonstrate thatL-PGDS levels in the senile NPH group were higher than in the presenileNPH group but significantly lower than in control and dementia groups(demential diseases other than NPH including vascular dementia,Alzheimer's disease and Parkinson's disease), indicating that NPH can bedifferentiated. L-PGDS levels in cerebrospinal fluid from both presenileand senile NPH groups were significantly lower than both control anddementia groups (p<0.005).

The neuron specific enolase (NSE) and S-100 protein levels in CSFindicative of brain parenchymal damage were also determined byradioimmunoassay and immunoradiometric assay, respectively. The resultsshowed that NSE levels in the CSF of each group increased in the order:presenile NPH group; senile NPH group; control group; and then dementiagroup. S-100 protein levels increased in the order: control group;dementia group; presenile NPH group; and then senile NPH group. NSElevels in cerebrospinal fluid showed no significant difference betweengroups except that presenile NPH group showed significantly lower levelsthan dementia group. S-100 protein levels in cerebrospinal fluid showedno significant difference between groups. Thus, L-PGDS was shown to bemore useful for differentiating demential diseases than NSE and S-100protein.

While the theory stated below remains speculative, the inventors inferthat L-PGDS production in NPH may decrease for the following reasons.L-PGDS is produced in arachnoid cells and secreted into spinal fluid,but arachnoid cells undergo dysfunction or decrease in number byinflammatory changes of the arachnoid membrane at the acute stage inpostsubarachnoid hemorrhage or postmeningitic NPH. This seems to resultin decreased L-PGDS production as compared with controls. L-PGDSproduction does not seem to be decreased in other demential diseases ascompared with NPH because dysfunction of the arachnoid membrane orcirculatory failure of spinal fluid is not found in these diseases.

Methods of the present invention can be used to differentiate reliablyand with a minimum of stress to a patient NPH that was not detectable orthat was difficult to detect reliably using the various conventionaltest means available, thus allowing early determination of suitabilityof a patient for surgery. Differentiation can be more reliable bycombining differentiation methods of the present invention with otherdiagnostic methods.

The present invention also provides a kit for differentiating ademential disease comprising an antibody specific to humanlipocalin-type prostaglandin D synthase (L-PGDS). Suitable antibodiesspecific to L-PGDS include monoclonal or polyclonal antibodies specificto L-PGDS, preferably various L-PGDS-specific monoclonal antibodiesmentioned above.

When an enzyme is used as a label for detection, the kit of the presentinvention can comprise the following component reagents:

(1) an enzyme-labelled monoclonal antibody, and

(2) a substrate solution

A variant of said kit using sandwich ELISA can comprise the followingreagents:

(1) a monoclonal antibody,

(2) an enzyme-labelled monoclonal or polyclonal antibody, and

(3) a substrate solution.

Another variant of said kit using biotin-avidin assay can comprise thefollowing reagents:

(1) a biotinylated monoclonal antibody,

(2) an enzyme-labelled avidin or streptavidin, and

(3) a substrate solution.

Another variant of said kit using sandwich ELISA and biotin-avidin assaycan comprise the following reagents:

(1) a monoclonal antibody,

(2) a biotinylated monoclonal or polyclonal antibody,

(3) an enzyme-labelled avidin or streptavidin, and

(4) a substrate solution.

For details of the process for preparing monoclonal and polyclonalantibodies used in the present invention see WO97/16461.

The following examples further illustrate the present invention without,however, limiting the scope of the invention thereto.

EXAMPLES Reference Example Determination of L-PGDS Levels

L-PGDS levels in body fluid samples were determined by sandwich ELISA.

(1) In order to prepare a standard curve, 300 μl/well of an anti-L-PGDSmonoclonal antibody (clone: 7F5) capable of binding L-PGDS diluted to4.4 μg/ml in 50 mM carbonate buffer (pH 9.6) was first added to a96-well microtiter plate and immobilized by allowing the plate to standovernight at 4° C. This plate was washed with phosphate-bufferedphysiological saline (pH 7.4, hereinafter referred to as PBS) threetimes, and then blocked by incubation with 300 μl/well of PBS containing0.2% casein (pH 7.4, hereinafter referred to as blocking solution) at30° C. for 90 minutes. Then, the blocked plate was washed with PBScontaining 0.05% Tween 20 (T-PBS) three times and then incubated at 30°C. for 90 minutes with 100 μl/well of a standard L-PGDS solution(prepared by serial dilution of L-PGDS purified from CSF in blockingsolution). After reaction, the plate was washed with T-PBS three timesand incubated at 30° C. for 90 minutes with 100 μl/well of horseradishperoxidase-labelled anti-PGDS monoclonal antibody (clone: 1B7) dilutedto 0.5 μg/ml in blocking solution. The plate was washed with T-PBS threetimes and then incubated at 30° C. for 30 minutes with 100 μl/well of acolor developing solution (ABTS solution available fromBoehringer-Mannheim), and then the reaction was stopped by adding 100μl/well of a quenching solution (1.5% oxalic acid) and shaking on aplate mixer. The difference between the absorbances at 405 nm and 490 nm(A405 nm-A490 nm) was determined with a commercially available platereader (Catalog # SK601 made by Seikagakusha) to prepare a standardcurve.

The monoclonal antibodies used in the sandwich ELISA described above(clones: 1B7 and 7F5) were obtained by injecting 1.0 ml of pristane intothe abdominal cavity of a mouse, implanting 1×10⁸ cells of a cell lineproducing each antibody into the abdominal cavity of the mouse after 2weeks, collecting the ascites after further 2 weeks and purifying theascites by protein A affinity column chromatography (3-10 mg/ml). Thecell lines producing the monoclonal antibodies described above areidentified with the designations of the respective monoclonal antibodiesand have been internationally deposited under Budapest Treaty with theNational Institute of Bioscience and Human-Technology of the Agency ofIndustrial Science and Technology (residing at 1-3, Higashi 1-Chome,Tsukuba-city, Ibaraki-prefecture, Japan) under accession number FERMBP-5709 (originally deposited on Sep. 21, 1996) for 1B7 and accessionnumber FERM BP-5711 (originally deposited on Jun. 6, 1996) for 7F5.

(2) L-PGDS levels in samples were determined by the sandwich ELISAdescribed above after diluting the samples in blocking solution asappropriate.

Example 1 Determination of L-PGDS Levels in the Cerebrospinal Fluid ofControl Subjects and Patients with Postsubarachnoid Hemorrhage NPH andIdiopathic NPH, Cerebrovascular Dementia, Parkinson's Disease andAlzheimer's Disease

L-PGDS levels in the CSF collected from the lumbar vertebra of 6controls with no abnormality except for intraorbital hematoma orheadache, 12 cases of postsubarachnoid hemorrhage NPH, 1 case ofidiopathic NPH, 3 cases of cerebrovascular dementia, 12 cases of,Parkinson's disease and 1 case of Alzheimer's disease were determined.

L-PGDS levels in the CSF of each group were 14.58±1.67 (μg/ml, mean±SD)in control group, 8.51±3.20 in postsubarachnoid hemorrhage NPH, 8.12 inidiopathic NPH, 26.45±5.67 in vascular dementia, 44.46±29.08 inParkinson's disease and 43.02 in Alzheimer's disease. A test ofsignificance between groups showed significant differences betweenpostsubarachnoid hemorrhage NPH and control groups, postsubarachnoidhemorrhage NPH and vascular dementia groups, and postsubarachnoidhemorrhage NPH and Parkinson's disease groups (p<0.005, p<0.0001,p<0.005). No significant difference was found between control group andvascular dementia or Parkinson's disease group (FIG. 1).

Example 2 Determination of L-PGDS Levels in the Cerebrospinal Fluid ofControl Subjects and Patients with Presenile NPH and Senile NPH andDementia

L-PGDS levels in the CSF collected from the lumbar vertebra of controls(8 cases) and patients with presenile NPH (under 70 years of age: 7cases), senile NPH (at or more than 70 years of age: 8 cases), dementiaexcluding NPH (a total of 7 cases including 4 cases of cerebrovasculardementia, 1 case of Parkinson's disease, 2 cases of Alzheimer's disease)were determined.

As a result, L-PGDS levels in the CSF of each group were 15.70±2.97(μg/ml, mean±SD) in control group, 7.05±1.69 in presenile NPH group,10.04±3.73 in senile NPH group and 19.14±4.34 in dementia group. Asshown in FIG. 2, L-PGDS levels in the cerebrospinal fluid of bothpresenile NPH and senile NPH groups were significantly lower as comparedwith both control and dementia groups (p<0.005). (In the figure, y-NPHmeans presenile NPH, e-NPH means senile NPH and dementia means dementiaexcluding NPH).

Example 3 Determination of Neuron Specific Enolase (NSE) and S-100Protein Levels in the Cerebrospinal Fluid of Control Subjects andPatients with Presenile NPH and Senile NPH and Dementia

CSF samples collected from individuals of each group were centrifuged(1500 g, 10 minutes) to give 0.5 ml of cell-free supernatants, whichwere stored at −20° C. and assayed for neuron specific enolase (NSE) andS-100 protein levels indicative of brain parenchymal damage. NSE wasdetermined by radioimmunoassay using an NSE assay kit (Eiken, Tokyo,Japan) and S-100 protein was determined with S-100 immunoradiometricassay kit (Sangtec Medical, Sweden).

As a result, NSE levels in the CSF of each group were 9.90±3.19 (ng/ml,mean±SD) in control group, 6.13±3.01 in presenile NPH group, 7.37±5.42in senile NPH group and 11.86±4.38 in dementia group (FIG. 3). On theother hand, S-100 protein levels were 0.98±0.38 (μg/ml, mean±SD) incontrol group, 3.27±3.04 in presenile NPH group, 3.68±3.61 in senile NPHgroup and 1.94±1.06 in dementia group (FIG. 4). NSE levels incerebrospinal fluid showed no significant difference between groupsexcept that presenile NPH group showed significantly lower levels thandementia group. S-100 protein levels in cerebrospinal fluid showed nosignificant difference between groups.

The foregoing results showed that NPH, which could not be detected byother indicators, can be detected and differentiated from otherdemential diseases by determining L-PGDS levels in CSF in comparisonwith normal levels.

1. A method to facilitate diagnosis of normal pressure hydrocephalus(NPH), comprising: measuring the concentration of human lipocalin-typeprostaglandin D synthase (L-PGDS) in a test sample of cerebrospinalfluid collected from a test subject, and comparing the concentration ofthe human L-PGDS in the test sample with normal levels of human L-PGDS,said normal levels being determined from control subjects not sufferingfrom NPH, wherein a significantly lower concentration of human L-PGDS insaid test sample taken from the test subject, compared to normal levelsof human L-PGDS is an indication that the test subject has NPH.
 2. Themethod of claim 1, wherein the normal pressure hydrocephalus issymptomatic normal pressure hydrocephalus or idiopathic normal pressurehydrocephalus.
 3. The method of claim 1 or 2, wherein the concentrationof human lipocalin-type prostaglandin D synthase is measured by animmunoassay.
 4. The method of claim 3, wherein the concentration ofhuman lipocalin-type prostaglandin D synthase is measured by sandwichELISA using a monoclonal antibody specific to human lipocalin-typeprostaglandin D synthase.